DESCRIPTION (from the application): The objective of this research proposal is to begin to dissect the complex series of molecular events involved in osteoblast differentiation by identifying and characterizing growth and transcription factors whose induction results in, or is associated with this process. Preliminary studies have identified activin betaA and follistatin as genes induced during this model of osteoblast differentiation. The role of these in osteoblast differentiation and endochondral bone formation will be defined by in situ hybridization of embryonic mouse limb bones. The effects of treatment with recombinant activin betaA and follistatin on osteoblast differentiation and endochondral bone formation will be investigated in MLB13MYC clone 17 cells as well as cultured whole embryonic mouse limbs. Monitoring of treatment effects will be performed by histological analysis, in situ hybridization and northern analysis for markers of bone and cartilage. Parallel experiments in activin beta A knockout limbs and follistatin knockout limbs will be performed to elucidate the role of each molecule independently of the other. A prechondroblastic cell line (MLB13MYC clone 17) which acquires markers of the osteoblast phenotype, including expression of osteocalcin, in response to recombinant human bone morphogenetic protein-2 (rhBMP-2) will be used for differential display reverse transcription polymerase chain reaction (ddPCR). Comparison of mRNA from rhBMP-2 treated and untreated MLB13MYC clone 17 cells will identify PCR products induced by rhBMP-2 treatment. These will be further characterized by northern analysis, sequencing, and comparing to GenBank data base sequences. Those sequences with homology or identity to growth factors or transcription factors will be used for in situ hybridization on day 16.5 embryonic mouse limbs to define their spatial and temporal expression during endochondral bone development. Differentially expressed products without data base match will either be extended using rapid amplification of cDNA ends (RACE) to gain more sequence information or used (in future projects) to screen a cDNA library.